© 2015. Published by The Company of Biologists Ltd | Biology Open (2015) 4, 1163-1179 doi:10.1242/bio.012468 RESEARCH ARTICLE The evolution of the dystroglycan complex, a major mediator of muscle integrity Josephine C. Adams1,‡ and Andrea Brancaccio2,*,‡,§ ABSTRACT of a plethora of interacting glycoproteins, predominantly laminin, Basement membrane (BM) extracellular matrices are crucial for the collagen IV, perlecan and nidogen, that are secreted from coordination of different tissue layers. A matrix adhesion receptor that surrounding cellular layers and assembled into dense, thin, is important for BM function and stability in many mammalian tissues is proteinaceous sheets by cell-ECM and extracellular interactions the dystroglycan (DG) complex. This comprises the non-covalently- (Breitkreutz et al., 2013). Basement membranes function to separate associated extracellular α-DG, that interacts with laminin in the BM, epithelial, endothelial, muscle, or neuronal cell layers from other and the transmembrane β-DG, that interacts principally with dystrophin tissue layers and compartments (Halfter et al., 2013). Laminins are to connect to the actin cytoskeleton. Mutations in dystrophin, DG, or amongst the most prominent ECM components of basement several enzymes that glycosylate α-DG underlie severe forms of membranes and are essential for the assembly of the molecular human muscular dystrophy. Nonwithstanding the pathophysiological networks that underlie basement membrane structure (Hohenester importance of the DG complex and its fundamental interest as a non- and Yurchenco, 2013). Laminin molecules are αβγ heterotrimers integrin system of cell-ECM adhesion, the evolution of DG and its and the C-terminal end of the laminin α-subunit is pivotal in the interacting proteins is not understood. We analysed the phylogenetic entire network due to its series of laminin globular (LG) domains, distribution of DG, its proximal binding partners and key processing which harbour binding sites for multiple cellular receptors including enzymes in extant metazoan and relevant outgroups. We identify that integrins, syndecans, Lutheran blood group glycoprotein and DG originated after the divergence of ctenophores from porifera dystroglycan (DG) (Durbeej, 2010). The latter represents the and eumetazoa. The C-terminal half of the DG core protein is major, non-integrin, laminin receptor in mammals, due to its wide highly-conserved, yet the N-terminal region, that includes the laminin- tissue distribution and important pathophysiological roles (Winder, binding region, has undergone major lineage-specific divergences. 2001). Phylogenetic analysis based on the C-terminal IG2_MAT_NU region The mature form of DG is a type I transmembrane protein identified three distinct clades corresponding to deuterostomes, composed of two non-covalently interacting subunits: α-DG, which arthropods, and mollusks/early-diverging metazoans. Whereas is extracellular and highly glycosylated, and β-DG, which the glycosyltransferases that modify α-DG are also present in contains the transmembrane and cytoplasmic domains (Ibraghimov- choanoflagellates, the DG-binding proteins dystrophin and laminin Beskrovnaya et al., 1992) (Fig. 1A). The two subunits are produced originated at the base of the metazoa, and DG-associated sarcoglycan from a single transcript, with α-DG liberated in the endoplasmic is restricted to cnidarians and bilaterians. These findings implicate reticulum by an unknown enzyme. During trafficking through extensive functional diversification of DG within invertebrate lineages the Golgi apparatus, crucial glycosyltransferases, SGK196, an and identify the laminin-DG-dystrophin axis as a conserved adhesion O-mannose kinase (Yoshida-Moriguchi et al., 2013), and B4GAT1, system that evolved subsequent to integrin-ECM adhesion, likely to a glucuronyltransferase (Praissman et al., 2014; Willer et al., 2014), enhance the functional complexity of cell-BM interactions in early act in a concerted and chronologically regulated fashion to modify metazoans. the α-DG core protein. These modifications are essential for the downstream enzymatic action of like-acetylglucosaminyltransferase KEY WORDS: Dystroglycan, Protein domain analysis, Metazoa, (LARGE), which adds a repeating disaccharide unit [-α3-GlcA-β3- Multicellularity, Basement membrane, Dystroglycanopathy Xyl-] to a mucin-like, central region of α-DG. These carbohydrate decorations of α-DG are important for the calcium-dependent, high- INTRODUCTION affinity binding of α-DG to the LG4 and LG5 domains of laminin α- Basement membranes are a specialised and structurally distinct subunits (Barresi and Campbell, 2006; Hara et al., 2011a; Tisi et al., form of metazoan extracellular matrix (ECM). They are composed 2000; Harrison et al., 2007) as well as to a number of other LG domain-containing extracellular ligands of α-DG, including agrin, perlecan and neurexin (see Sciandra et al., 2013 and references 1School of Biochemistry, University of Bristol, Biomedical Sciences Building, β University Walk, Bristol BS8 1TD, UK. 2Istituto di Chimica del Riconoscimento therein). Intracellularly, the extreme C-terminus of -DG binds to Molecolare, CNR, Istituto di Biochimica e Biochimica Clinica, UniversitàCattolica the cysteine-rich, C-terminal domain of the actin-binding protein, del Sacro Cuore, Largo Francesco Vito 1, Roma 00168, Italy. dystrophin, ensuring a connection to the F-actin cytoskeleton (Jung *Present address: School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK. et al., 1995). The C-terminus also interacts with the dystrophin- ‡ Both authors contributed equally to this work related protein, utrophin, which is predominantly expressed at neuro-muscular junctions (Ishikawa-Sakurai et al., 2004). §Author for correspondence ([email protected]) The bridging function of the mature DG complex between This is an Open Access article distributed under the terms of the Creative Commons Attribution external basement membranes and the intracellular F-actin License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. cytoskeleton is considered to be a major determinant of sarcolemma and fibre stability in skeletal muscle (Holmberg and Received 28 April 2015; Accepted 19 July 2015 Durbeej, 2013), where the DG complex functions as part of the Biology Open 1163 RESEARCH ARTICLE Biology Open (2015) 4, 1163-1179 doi:10.1242/bio.012468 Fig. 1. Domain architectures of dystroglycans from different animal phyla. (A) The DG typical of vertebrates Callorhincus milii (elephant shark), Lethenteron japonicum (Cyclostomata), (Strongylocentrotus purpuratus (Echinoderma) and an annelid, Capitella teleta. (B-J) domain architectures of DG identified in (B) Urochordata (Ciona intestinalis) and Cephalochordata (Branchiostoma lanceolatum), (C) Hemichordata (Saccoglossus kowalevskii), (D) Mollusca, (Gastropods Lottia gigantea and Aplysia californica, Bivalve, Crassostrea gigas and cephalopod, Octopus vulgaris), (E) arthropod classes (Insecta and Hymenoptera, Camponotus floridanus and others) (see also supplementary material Fig. S1), (F) Nematoda (Caenorhabditis elegans and Caenorhabditis remanei), (G) Cnidaria, Hydra magnipapillata, (H) Cnidaria, Nematostella vectensis (see also supplementary material Fig. S2), (I) Placozoa (Trichoplax adhaerens), (J) Porifera (homoscleromorph Oscarella carmela) (see also supplementary material Fig. S3). Expansions of the IG2_MAT_NU module are indicated in C, E and I (2×) and in H (6×). Black arrowheads indicate the furin cleavage site. Red arrowheads indicate the Gly-Ser α/β maturation site. SP, signal peptide; IG1 and IG2, immunoglobulin-like domains; S6, S6-like domain; βBS, β-subunit binding site on the IG2 domain; MAT, C-terminal region of α-dystroglycan upstream of the Gly-Ser maturation site; NU, natively unfolded region that forms the N-terminal region of the ectodomain of β-dystroglycan; TM, transmembrane; cyto, cytoplasmic domain; DBS, dystrophin-binding site. The SP is reported as a black box if complete, or a white box if partial. Dotted lines indicate protein sequences that are incomplete at the N-terminal end. Dotted boxes around the IG domains of Urochordata (B) or the S6 domain of nematodes (F) indicate the divergence of these domains (less than 20% sequence identity). The dotted box for NU in H. magnipapillata DG (G) indicates the presence of two deletions in this region. The white box within the cytoplasmic domain of N. vectensis DG (H) indicates the presence of an insertion. In T. adhaerens DG (I), no DBS was detected (white box). Diagrams are not to scale. Accession codes and other details are in Table 2. dystrophin-glycoprotein complex (DGC), a multi-protein complex transmembranous core of the complex includes sarcoglycans and originally identified in rabbit skeletal muscle (Ervasti and the tetraspanin-like protein, sarcospan, as well as DG (Ervasti and Campbell, 1991; Ibraghimov-Beskrovnaya et al., 1992). The Campbell, 1993). It is now appreciated that the DGC has an Biology Open 1164 RESEARCH ARTICLE Biology Open (2015) 4, 1163-1179 doi:10.1242/bio.012468 important role in the mechanical stability of multiple mammalian Table 1. Sequence relationship of dystroglycans from different phyla to tissues in addition to skeletal muscle, including the neuromuscular human dystroglycan. Sequences were identified from